Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell–enhancing target. In murine CD8+ T cell–therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.


Design and generation of screening libraries:
Viral vectors were constructed for both human and mouse T-cell screening.Human libraries were in a pLenti6-based lentiviral backbone that contained a human U6 promoter cassette for guide cloning, as well as a puromycin resistance marker and tagRFP fluorescent marker (pKSQ017) or a Thy1.1 selectable marker (pKSQ144).For retrovirus, the vector was a selfinactivating murine stem cell virus (MSCV) backbone containing the same human U6 promoter guide cloning cassette as well as a human CD2 marker for cell surface expression and bead enrichment of infected cells.The vectors used for TCR-Tg CRISPR screens additionally contained a library of randomly synthesized 12-mer barcodes with the nucleotide sequence BVHDBVHDBVHD (IUPAC mixed base codes).These random barcodes (theoretical diversity of 531,441 unique sequences), when randomly paired with the guide sequence during cloning, allow the ability to independently trace millions of unique infection events in immune cells.
Libraries were generated by searching for PAM sites (NGG) in the human and mouse genome that were expected to correspond to sgRNAs that would cut the coding sequence of genes of interest.sgRNAs were chosen for the library that were predicted to cut the genome in at most one site, while not overlapping with known high prevalence SNPs and having few (<10) closely related sites in the genome that could represent likely off-target sites.Several classes of controls were included, non-cutting controls, olfactory receptor cutting controls not expected to impact biology in immune cells, lethal shredder controls that cut the genome thousands of times, and (for human only) fingerprinting controls that cut high-prevalence SNPs in human essential genes and allowed internal identification of samples by donor.The final set of filtered sgRNAs were then synthesized on a microarray (Agilent oligo library synthesis) as oligonucleotides with adapters, PCR amplified, and cloned using the type IIs restriction enzyme BbsI into the final lentiviral or retroviral vector.
sgRNA library virus production Lentivirus production: Lentivirus was generated by lipid transfection of packaging plasmids into HEK293T cells.For a 10-layer CellStack (Corning), 300 million HEK239T cells were plated in 1 L of DMEM + 10% FBS.24 Hours later transfection was performed using 461 ug of sgRNA library plasmid, 231 ug of packaging plasmid psPax2 (Gag-Pol), and 115 ug of pMD2.G packaging plasmid (VSV-G).Plasmid DNA was added to 37.77 ml of room temperature OptiMEM media.2428 ul of TransIT transfection reagent (Mirus Bio) was added, mixed by vortexing, and incubated for 20 minutes before proceeding.OptiMEM+DNA+TransIT mixture was added to 1 liter of DMEM + 10% FBS.Media was aspirated from HEK293T cells and fresh media containing transfection mixture was added.24 hours after transfection the media was replaced 1 liter of UltraCulture media (Lonza) supplemented with L-glutamine.48 hours after transfection the supernatant was harvested from the cells, incubated for 1 hour at 37C in the presence of 50 units/ml benzonase, filtered using a 0.45 uM bottle top PES filter, and concentrated by tangential flow filtration on a spectrum labs KrosFlow mPES hollow fiber filter (100kD molecular weight cutoff).Virus was concentrated approximately 10-50x (depending on initial volumes being processed).
Retroviral Production: Retrovirus was generated by transient transfection of Phoenix-Eco retroviral packaging cells with library plasmid pools.5 million Phoenix-Eco cells were plated per 10 dish 24 hours before transfection in DMEM + 10% FBS.Transfection is carried out by mixing 10ug of library plasmid pool with 327 ul optiMEM media.21 ul of Mirus TransIT-293 transfection reagent is added to this mixture, vortexed briefly, and incubated for 20 mins.Transfection mixture is then added dropwise to previously plated phoenix cells and incubated.For infection of mouse CD8 cells, media is replaced with 5ml of RPMIc (RPMI + 10% HI FBS, 20 mM HEPES, 50 uM 2-mercaptoethanol).Virus is frozen and titered for subsequent large-scale transduction.
-T cell Transduction TIL: To transduce TIL5746, retronectin-coated non-TC-treated 6 well plates were washed with 1ml PBS, with 200ml of pKSQ017 Lib16 or RPL10a virus added and spun at 2000g for 2hrs.5x10 6 TIL/well in 1ml complete XVIVO-15 media were added to each well (total volume 1.2ml) so that 200 x10e6 TIL5746 were transduced with Lib16, and 10x10e6 TIL5746 were transduced with sgRNAs targeting RPL10a.Plates were spun at 1000g for 10 minutes, and then transferred to 37C for 3 hours, followed by addition of 3ml of complete XVIVO-15 to all wells, and incubation at 37C overnight.
Mouse TCR-Tg cells: To transduce mouse CD8 TCR-Tg T cells isolated from spleens and activated with Dynabeads and 4ng/ml IL-2 on Day 0, non-TC-treated 6 well plates were coated in parallel with retronectin overnight at 4C.On Day 1, plates were washed with 1ml PBS and blocked with DPBS + 2% BSA for 30 minutes.Activated mouse TCR-Tg CD8 cells were then harvested and resuspended at a concentration of 1.5x10 6 /ml in thawed concentrated lentivirus or retrovirus supernatant with 2ng/ml IL-2 and 5mg/ml protamine sulfate.2ml of virus + CD8 mixture is pipetted in each well of the retronectin-coated 6-well plate, with plates centrifuged at 600g for 90 mins, and then transferred to a 37C TC incubator for 5 hours.2ml of cRPMI + 2ng/mL IL-2 was then added to each well and incubated overnight at 37C.Cells were harvested, washed, and re-suspended in cRPMI + IL2 for further expansion on the following day.The lentivirus (Thy1.1)and retroviruses (CD2) used for the OT1 and PMEL transductions, respectively, contain selectable markers for positive selection, with this selection step occurring on Day 3 following transduction.On Day 4, cells were cryopreserved for future use.

Cell Culture
B16-Ova, MC38-gp100 and A375-mOKT3 cells were cultured in DMEM (Gibco, Cat# 11885076) supplemented with 10% FBS (Gibco, Cat# 10082147) at 37°C in a 5% CO2 atmosphere.Cells were passaged two to three times per week to ensure confluency in the flask never surpassed 80%.Tumor cells were harvested in serum-free DMEM during exponential growth phase immediately prior to inoculation.Generation of sgOlf, sgPD-1 and sgSocs1 OT1s and PMELs: Spleens were harvested from OT1 or PMEL mice, placed in 5mL StemCell Buffer and dissociated in GentleMACS C-tubes using a GentleMACS octo dissociator.Following filtration and rinsing of cells, CD8 T cell were isolated using the EasySep TM Mouse CD8+ T Cell Isolation Kit (Stemcell, Cat# 19853) according to the manufacturer's instructions.Purified CD8 T cells were activated in the presence of 4ng/ml mouse rIL-2 with mouse CD3/CD28 Dynabeads.48 hours later, Dynabeads were removed, sgRNA / Cas9 RNPs prepared with sgRNA at 22mM and Cas9 at 15mM in IDTE, Buffer T and electroporation enhancer, and electroporated at 1700V, 20ms, 1pulse using a Neon transfection system (LifeTechnologies).Cells were expanded in the presence of 32ng/ml mouse rIL-2 for an additional 48 hours, harvested, and either transferred directly into recipient mice or cryopreserved in 90% FBS + 10% DMSO.

CRISPR Screens
Human in vitro TIL expansion screen: The 'Lib16' sgRNA library targeting 5,137 genes with 10sgRNAs/gene and 56,408 sgRNAs total, including controls, was cloned into pKSQ017 with semi-random barcodes (see Supplementary Methods for more information on library design and lentivirus production).Lib16 includes sgRNAs targeting genes involved in T cell function, all predicted cell surface receptors, all known immune-related genes, and all genes demonstrating expression in blood.Dissociated melanoma tumors from donor 110005746 were obtained from Conversant Bio and seeded at 1x5 6 cells/ml in TIL-CM (RPMI 1640 + 10% HI HS, 1X HEPES, GlutaMAX, 2-Mercaptoethanol, Pen/Strep, Gentamicin) containing 300ng/ml IL-2 in a 24 well tissue culture treated plate, and incubated at 37C.Every other day, 300ng/ml IL-2 was added to cell culture assuming consumption.On Day 5, suspension cells were harvested and re-seeded at 1.5x10e6/ml in TIL-CM combined 1:1 with XVIVO-15 and supplemented with 300ng/ml IL-2, with adherent cells observed to have disappeared from the wells.Between Days 7 through Day 22, cells were expanded in IL-2 with a 1:1 ratio of TIL-CM:XVIVO-15 media supplemented with 300ng/ml IL-2 added every other day in order to maintain cell density at 1e6 cells/ml.On Day 35, cellularity was evaluated, with composition found to be 66% CD8 + , and 32% CD4 + .TIL5746 were frozen on Day 35 in CS10 freezing medium at 1x10 8 cells/ml.On the first day of the screen (screen Day 0), 2x10 8 TIL were thawed, washed in 40ml XVIVO-15 media twice, and resuspended to 2x10 6 cells/ml in XVIVO-15 media containing 600ng/ml IL-2 and 1X DNase (Stemcell, Cat# 07900).In parallel, non-tissue culture treated 6 well plates were coated with 20ug/ml retronectin in PBS and incubated overnight in preparation for transduction.On Day 1, TIL were transduced with pKSQ017 lentivirus driving expression of Lib16 under control of the human U6 promoter, as well as driving expression of tagRFP under control of a UBC promoter.Semi-random barcodes were used to track individual clones.In parallel, an aliquot of TIL5746 were separately transduced with the KSQ041 lentivirus which drives expression of a sgRNA targeting the essential gene RPL10a under control of a human U6 promoter as well as mNeonGreen under control of a UBC promoter.Transduced cells were incubated in 6 well plates at 37C in XVIVO-15 overnight.On Day 2, TIL were combined from 6 well plates, spun at 300g for 5 minutes, supernatant removed, and TIL resuspended in complete XVIVO-15 media and rested overnight.On Day 3, transduced TIL5746 were electroporated with Cas9 mRNA (Trilink #L-7206, lot# T1COL01A) using an Amaxa 4D-Nucleofector unit and pulse code CA137 with Buffer P3 according to the manufacturer's instructions.Following electroporation, 80ml of pre-warmed XVIVO-15 media was added to each well, mixed, and wells combined and washed.TIL were re-suspended to 1x10 6 in complete XVIVO-15 media, and incubated overnight at 37C.On Day 4, 5x10 7 transduced TIL5746 were frozen to determine the input sgRNA Library distribution.The TIL Rapid Expansion Phase (REP) was initiated on 25x10e6 transduced and edited TIL with a 1:200 ratio of TIL to irradiated PBMCs derived from 5 pooled donors, 600ng/ml IL-2 and 30ng/ml OKT3 using XVIVO-15 media in a 5L Grex.Cells were incubated at 37C.On Days 8, 11 and 15, 2.5L of media was aspirated from the Grex and replaced with 2.5L complete XVIVO-15 media, with 3mgs of IL-2 added to the 5L Grex.On Day 17, TIL were counted, with 13.2x10 9 TIL5746 obtained, and analyzed for cellularity on a FACS, with 85.5% of the TIL CD8 + , and 12.9% of the TIL CD4 + .TIL were harvested for screen analysis by freezing three 5x10e7 cell pellets for gDNA preparations.The sgRNA distribution was compared between TIL harvested on Day 4 and Day 17.
In vivo Cas9-Tg x TCR-Tg CD8 T cell syngeneic tumor screens: The 'Lib30' sgRNA library targets 369 genes involved in T cell function with 10sgRNAs per gene and 3089 sgRNAs total, including controls, was cloned into pKSQ044.Lib31 sgRNA library targets 1004 predicted CSR genes with 8 sgRNAs/gene and 11,148 sgRNAs total, including controls, was also cloned into pKSQ044 (see Supplementary Methods for more information on library design and lentivirus production).Cas9-Tg x TCR-Tg OT1 or PMEL CD8 T cells were isolated from freshly harvested mouse spleens and dissociated using a GentleMACS (Miltenyi), and with CD8 T cells purified by negative selection (EasySep Mouse CD8+ T cell isolation kit).CD8s were then placed in T225 flasks at a concentration of 1M CD8s/ml and activated with CD3/CD28 Dynabeads and 2ng/ml mouse rIL-2.The following day, T cells were transduced wither either lentivirus (OT1 B16-Ova screen) or retrovirus (PMEL / MC38-gp100 screen).Dynabeads were removed the following day, with cells resuspended on cRPMI + 32ng/ml IL-2.On Day 4, transduced T cells were harvested and selected by positive selection using either Thy1.1 for OT1s or hCD2 for PMELs.5x10 6 Thy1.1 + Cas9-Tg x OT1 CD8 T cells or 7x10 6 hCD2 + Cas9-Tg x PMEL CD8 T cells were injected into tumorbearing mice i.v.via tail vein in 200mL PBS, with 10x10 6 CD8 + T cells saved to determine the sgRNA distribution of the input population of T cells.Group sizes were 7-8 mice per library.At the indicated time following adoptive transfer of cells, mice were euthanized and blood was harvested in EDTA tubes, and stored at -80C.Tumors, spleens, tumor draining and non-draining lymph nodes were harvested and processed further for extraction of CD8 T cells using CD8a Microbeads (Miltenyi, cat# 130-049-401) from the spleen and CD45 Microbeads (Miltenyi, cat# 130-052-301) from the tumor.Tumors were digested using the Miltenyi Tumor Dissociation Kit (Cat# 130-096-730) according to the manufacturer's instructions.Genomic DNA was isolated using the Qiamp Blood Midi and Maxi kids, according to the manufacturer's instructions.SOCS1 sgRNA Tiling Screen: Primary human CD8 + T cells were isolated from PBMCs, with ~300x10 6 CD8 T cells obtained by negative selection using a EasySep Human CD8+ T cell Isolation Kit (StemCell; Cat# 17953).T cells were activated with Immunocult Human CD3/CD28/CD2 T cell activators (Stemcell; Cat# 10990) in the presence of 10U human IL-2 in X-VIVO-15 media.On the following day, CD8 T cells were harvested, counted, and transduced with lentivirus expressing 134 sgRNAs targeting genomic positions across the full length of the SOCS1 gene as well as multi-cutter sgRNAs as depleting and Olfactory genes as neutral controls.Two days after transduction, T cells were electroporated with Cas9 mRNA (Trilink) and expanded inn 10U human IL-2 in X-VIVO-15 media for 10 additional days.Cells were then harvested; DNA was extracted and amplicons spanning the lentiviral genomic regions containing the sgRNA cassettes in the library were amplified by polymerase chain reaction (PCR) and sequenced by next-generation sequencing (NGS).
CRISPR Screen Pre-processing: Counts of sgRNA frequency were generated from FASTQ sequencing files by counting the number of occurrences of each 20nt sequence READ1.For libraries that employed a unique molecular identifier the frequency of each UMI was generated by tabulating the number of occurrences of each 12nt prefix of READ2.For libraries containing UMIs the resulting count tables were further cleaned by requiring 1) the UMI matches the mixing code used to synthesize the library 2) does not contain any ambiguous/uncalled bases 3) is not potential index-bleed (exactly matching a more abundant guide/UMI on the same sequencing run).To minimize the impact of singleton/low frequency clones a two-component mixture model was fit to the count frequency distribution and guides present in the low abundance component were dropped.
Analysis of in vitro TIL CRISPR Screens: To identify hits from the screen we calculated the foldenrichment of each candidate gene in an "end-point" relative to a "reference" sample.The reads from raw FASTQ files were counted, and the number reads representing each guide were tabulated.For each guide, a log-ratio between the screen end-point and the reference sample was calculated.To compute a robust gene-level enrichment score, guide level scores were aggregated by taking the median enrichment score across all guides.We assigned each guide a conservative p-value equal to the percentile of its logFC among all guides in the library.To calculate a p-value for gene enrichment individual guide-level p-values were combined using Fisher's method.The resulting p-values were adjusted for false discovery rates by the Benjamini-Hochberg method.To standardize (Z-score) the resulting scores, logFC scores were centered and scaled by subtracting the median enrichment score and dividing by the median absolute deviation to calculate an overall effect size for each gene.
Analysis of in vivo B16-OVA and MC38-gp100 CRISPR Screens: MAGeCK-mle(Version 0.5.9.3) was used to perform CRISPR screen analysis.Genes with guide numbers fewer than 5 were removed from the analysis.The number of unique UMI for each sgRNA were summarized as input clone counts.Clone counts were organized from all in-vivo tumor samples and input samples into a count matrix.Due to the large number of zero counts in the matrix, an altered RLE normalization was performed on raw count matrix by using geometric mean of gene and size factor of one sample calculated by only non-zero counts.Normalized count matrix was used as MAGeCK-mle input.Design matrix was composed of two columns: input column with all "1" as baseline level, and tumor column with only tumor samples as "1" and the rest samples as "0".Gene level beta values from MAGeCK-mle output were used to evaluate the effect size of knocking out the corresponding gene.

Assessment of Editing Efficiency
Amplicon Sequencing (Amp-Seq): To assess on-and off-target editing efficiencies in sgRNA/Cas9 RNP edited T cells, gDNA was extracted from edited T cells using the XTRACT16+ following the manufacturer's protocol (Autogen, cat# KX110-96).Following the extraction of gDNA, a two-step library preparation method was performed.First step PCR consisted of a multiplex PCR reaction amplifying target sites, followed by a second step PCR adding on Illumina adapters consisting of indexing to allow for multiplexed NGS.Editing efficiency was then assessed by aligning reads to the regions of interest and the fraction of indel reads was calculated to yield a cutting score.SOCS1 protein Wes: TIL or KSQ-001 cells were stimulated by ImmunoCult Human CD3/CD28/CD2 T cell activator (STEMCELL, Cat#10970) following manufacturer's protocol overnight in REP TIL media.Cell pellets were collected, lysed by RIPA buffer (Sigma, Cat#R0278) containing Protease and Phosphatase Inhibitor (ThermoFisher, Cat# A32961).Lysates were centrifuged at 21,000g for 10min at 4°C; supernatant was subjected to BCA assay (Pierce, Cat# 23227) for protein quantification.SOCS1 (Cell Signaling, Cat# 68631S) and loading control, Vinculin (Cell Signaling, Cat# 13901S) were detected via Wes instrument (ProteinSimple) following manufacturer's protocol.

scRNA-Seq
Droplet-base 5' single-cell RNA sequencing (scRNA-Seq) was performed by the 10x Genomics platform and libraries were prepared by the Chromium Single Cell 5' Reagent kit according to the manufacturer's protocol (10x Genomics, CA, USA).The Cell Ranger (version 6.0.1) was used for gene expression quantification, TCR sequence assembly, and cell identification.Cell level quality control was performed using the function quickPerCellQC() from scater (version 1.18.6).Per-cell size-factor normalization was performed using the computeSumFactors() function from scran (version 1.18.7).Cell lineages were annotated automatically using clustifyr (version 1.2.0) and the Haemosphere mouse RNA-seq database(58).T cells were isolated by computational gating based on expression of Cd3d, Cd3e, and Cd3g.Seurat (version 4.1.0)was used to identify clusters and perform differential gene expression analysis.Differentially expressed genes were identified using the Wilcoxon Rank Sum test.The hypergeometric test was performed to assess treatment group enrichment in each of the identified clusters.ProjecTILs (version 2.0) was used to map our data onto scRNA-Seq data from Miller et al (45).Fgsea (version 1.26.0) was used to perform GSEA on the CD8 expressing T cells using Tex gene signatures from Miller et al. and Beltra(45,59).GSVA (version 1.48.2) was used to perform GSVA on the CD8 expressing T cells using Tex gene signatures from Miller et al. and Beltra et al.The extent of clonal expansion per cluster was quantified using the StartracDiversity() function from scRepertoire (version 1.10.0).OT1 T cells were identified by searching the TCR repertoire for cells containing both the OT1 CDR3 Tcra amino acid sequence (CAASDNYQLIW) and the OT1 CD3R Tcrb amino acid sequence (CASSRANYEQYF).

5ʹ-Phos-G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T-3ʹ and 5ʹ-Phos-A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C-3ʹ.
Each oligonucleotide is phosphorylated at the 5' end and includes phosphorothioate bonds (noted by asterisks).For GUIDE-Seq, primary T cells were electroporated with RNP complexes as described above with the addition of 200pmol of the annealed dsODN.The single-tail Y adapter was formed after annealing the following DNA strands: CTACAAGAGCGGTGAGT and 5ʹ-Phos-CTCACCGCTCTTGTAGSNNNNNNNNCTGTCTCTTATACACATCTCCGAG*C.The tail of the adapter includes a unique molecular identifier (indicated by the sequence SNNNNNNNN).Double-stranded genomic DNA was quantified by the Qubit Broad Range Assay (Cat # Q32853) and 1 μg of DNA from each sample was used for library preparation using the NEBNext Ultra II FS DNA Library Prep kit (Cat # E6177L).Genomic DNA was fragmented to 300-700bp (37°C incubation for 10 minutes) and 5 μl of 10 μM annealed adapter was used in the subsequent adapter ligation step.Purified adapter ligated DNA was used for two separate touch-down PCR enrichment reactions of dsODN containing fragments (plus and minus strand orientations each paired with a primer binding to the Y adapter).These primers are: Minus strand dsODN primer -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNATACCGTTATTAACATATGACAACTCAATTAAA *C, Plus strand dsODN primer -TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNGTTTAATT GAGTTGTCATATGTTAATAACGGTA*T, and Adapter primer -GTCTCGTGGGCTCGGAGATGTGTATA AGAGACA*G.
The double-strand DNA products from these reactions were quantified and 5ng from each sample's plus and minus reactions were pooled for indexing PCR using primers from the Nextera DNA CD Indexes 96 well plate (Cat # 20018708).Final PCR products were quantified and library distribution was measured by D1000 TapeStation (Cat # 5067-5582 and 5067-5583).All samples were pooled by equal mass and purified by 1.2X SPRI bead clean-up.The concentration of the final pool was quantified by Kapa Illumina Library quantification kit (Cat #KK4923).Final pools were loaded onto the NextSeq with at least 10% PhiX spike-in to ensure sequence diversity.

Engineering of high-and low-affinity A375-mOKT3 cells
The A375-mOKT3 cell line was generated by infecting A375 melanoma cells with a lentiviral vector encoding a blasticidin resistance gene and either a high affinity (parental OKT3 sequence) or low affinity version (two amino acid substitutions to reduce affinity for CD3 ~1000 fold) of the mOKT3 protein.(2)Individual clones were selected and tested for their ability to stimulate TIL in vitro.

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Supplementary Methods o Design and generation of screening libraries o sgRNA library virus production o T cell transduction o Cell Culture § Culture of syngeneic tumor cells § Generation of sgOlf, sgPD-1, and sgSocs1 OT1 and PMELs § Human TIL § IFNg release § AICD § TIL / Tumor co-culture § pSTAT Assay § A375-mOKT3 spheroid killing assay o CRISPR Screens o Assessment of engineering efficiency by amplicon sequencing and WES o scRNA-Seq o GUIDE-seq o Engineering of high-and low-affinity A375-mOKT3 cells o sgRNA sequences o Amplicon sequencing primers for on-and off-target editing assessment o FACS o Antibody Table • Supplemental Data o Supplemental Data 1: Human TIL screen QC o Supplemental Data 2: OT1 / B16-Ova screen QC o Supplemental Data 3: PMEL / MC38-gp100 screen QC • Supplementary Figures oFigure S1: Development of a syngeneic tumor models sensitive and insensitive to inactivation of PD-1 in adoptively transferred TCR-Tg CD8 T cells o Figure S2: Inactivation of SOCS1 enhances the in vivo anti-tumor potency of CD8 T cells and drives the accumulation of CD44 + CD62L + Tcm memory cells in blood o Figure S3: FACS analysis of innate cell subsets from sgOlf, sgSocs1 and sgPD-1 OT1s from the TME 7 days following transfer o Figure S4: scRNA-Seq of sgOlf, sgSocs1 and sgPD-1 OT1s from the TME 7 days following transfer o Figure S5: Identification of the genomic locations of u728 sgRNA off-target cut sites o Figure S6: Characterization of KSQ-001 o Figure S7: Sensitivity of KSQ-001, TIL, and sgSOCS1-edited CD3 + T cells to IL-2 and IL-15

Figure S2 :
Figure S2: Inactivation of SOCS1 enhances the in vivo anti-tumor potency of CD8 T cells and drives the accumulation of CD44 + CD62L + Tcm memory cells in blood C57BL/6 mice bearing B16-Ova tumors cells at a median size of 100mm 3 on the flank were treated with SOCS1 (sgSocs1) or OLF1 (sgOlf) OT1 CD8 T cells as indicated.Editing efficiencies for target genes were 91% for sgSocs1 and 71% for sgOlf.(a) Tumor growth curves over time are depicted.(b) The frequency of Va2 + Vb5.1 + OT1s present in the peripheral blood of mice receiving a dose of 41x10 6 OT1s is depicted 21 days following transfer.(c) the frequency of CD44 + CD62L + Tcm and CD44 + CD62L -Tem OT1s were quantified in mice receiving 41x10 6 OT1s 21 days following transfer.Each dot represents an individual mouse, with **** = p value < 0.0001; and * = p value < 0.05 by unpaired, two-tailed Student's t test between the indicated comparator groups.

Figure S3 :
Figure S2 Figure S4: scRNA-Seq of sgOlf, sgSocs1 and sgPD-1 OT1s from the TME 7 days following transfer.(a) Distribution of general lymphocytes populations from CD45+ cells from the TME.Cells were annotated computationally, with the Haemosphere mouse RNA-Seq database(3) used to verify the predominate lymphocyte clusters.(b) General lymphocyte population frequency per treatment group.(c) Cluster-wise cell counts per treatment group.(d) Top 10 DEGs (by avgLog2FC) per cluster.Significance was determined using the Wilcoxon Rank Sum test and genes with an adjusted p value < 0.1 were considered significant.(e) UpSet plot of DEG counts between treatment groups within the OT1 population.(f) Heatmap of median GSVA scores of Miller et al.(3) Tex gene signatures and Beltra et al.(4) Tex gene signatures per Tex cluster by treatment group.(g) Boxplot of GSVA scores of five TCR activation-related gene signatures from Reactome by Tex cluster.(h) Barplot of T cell cluster frequency by treatment group.(i) UpSet plots of DEG counts between treatment groups per Tex cluster.(j) Volcano plots depicting DEGs

Figure S5 :
Figure S5: Identification of the genomic locations of u728 sgRNA off-target cut sites (a) GUIDE-Seq was used to quantify and characterize the frequency of on-and off-target cut sites in primary human T cells.Two sgRNAs targeting CCR5 (R-30 and R-25) with high off-target editing frequencies were included as benchmarks, with the SOCS1 on-target editing efficiency depicted in blue, and the cumulative editing efficiency of off-target loci depicted in red for the u728, kipc and qd5u sgRNAs.(b) Amp-Seq confirmation of on-and off-target editing for the u728 sgRNA in human TIL including 22 potential off-target sites identified in (a).The u728 sgRNA achieved a median of 96% editing of the SOCS1 gene.Off-Target-01 confirmed as reaching statistically significant levels of 0.3%.(d) The genomic location of the Off-Target-01 site is in an intragenic region on Chromosome 7 with no known regulatory regions.

Figure S6 :
Figure S6: Characterization of KSQ-001 TIL and KSQ-001 were manufactured.(a) Fold expansion of TIL and KSQ-001 cells over 14 days of REP.(b) Following manufacture and cryopreservation, TIL and KSQ-001 were thawed and viability assessed by AOPI staining, with each dot reflecting an independent paired donor (c) To evaluate the ability of KSQ-001 to undergo activation-induced cell death (AICD) following activation, cryopreserved TIL and KSQ-001 cells were thawed, rested overnight, and activated with the indicated concentration of CD3 tetramer for 18 hours.The AICD profile of CD4 and CD8 within TIL and KSQ-001 was measured by FACS analysis of Caspase-3 staining.(d) The frequency of donor-paired CD4 and CD8 cells within KSQ-001 and TIL is depicted.Each dot reflects an independent donor.(e) The frequency of central memory cells (CCR7 + , CD45RO + ) and effector

Figure S7 :
Figure S7: Sensitivity of KSQ-001, TIL, and sgSOCS1-edited CD3 + T cells to IL-2 and IL-15 The sensitivity of KSQ-001 and TIL to IL-2 and IL-15 through STAT5 phosphorylation (pSTAT5) was evaluated.Following REP and cryopreservation, donor-paired TIL and KSQ-001 were thawed, rested, and activated with the indicated cytokine.(a) pSTAT5 signals between TIL and KSQ-001 following IL-2 activation.(b) pSTAT5 signals between TIL and KSQ-001 following IL-15 activation.(c) CD3 + Pan-T cells were activated and edited sgSOCS1, with sensitivity to IL-2 through pSTAT signals evaluated at 7 days following activation in comparison to unengineered cells.(d) Same experiment as in (c), but with pSTAT5 sensitivity to IL-2 evaluated at 13 days following initial activation.Statistical significance: * = p < 0.05 using a Student's t test between the indicated groups.ns = not significant